Yb modulates the divisions of both germline and somatic stem cells through piwi- and hh-mediated mechanisms in the Drosophila ovary.

The coordinated division of distinctive types of stem cells within an organ is crucial for organogenesis and homeostasis. Here we show genetic interactions among fs(1)Yb (Yb), piwi, and hedgehog (hh) that regulate the division of both germline stem cells (GSCs) and somatic stem cells (SSCs), the two constituent stem cell populations of the Drosophila ovary. Yb is required for both GSC and SSC divisions; loss of Yb function eliminates GSCs and reduces SSC division, while Yb overexpression increases GSC number and causes SSC overproliferation. We also show that Yb acts via the piwi- and hh-mediated signaling pathways that emanate from the same signaling cells to control GSC and SSC division, respectively. hh signaling also has a minor effect in GSC division.     

Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation

The transition from a Drosophila ovarian germline stem cell (GSC) to its differentiated daughter cell, the cystoblast, is controlled by both niche signals and intrinsic factors. piwi and pumilio (pum) are essential for GSC self-renewal, whereas bag-of-marbles (bam) is required for cystoblast differentiation. We demonstrate that Piwi and Bam proteins are expressed independently of each other in reciprocal patterns in GSCs and cystoblasts. However, overexpression of either one antagonizes the other in these cells. Furthermore, piwi;bam double mutants phenocopy the bam mutant. This epistasis reflects the niche signaling function of piwi because depleting piwi from niche cells in bam mutant ovaries also phenocopies bam mutants. Thus, bam is epistatic to niche Piwi, but not germline Piwi function. Despite this, bam- ovaries lacking germline Piwi contain approximately 4-fold fewer germ cells than bam- ovaries, consistent with the role of germline Piwi in promoting GSC mitosis by 4-fold. Finally, pum is epistatic to bam, indicating that niche Piwi does not regulate Bam-C through Pum. We propose that niche Piwi maintains GSCs by repressing bam expression in GSCs, which consequently prevents Bam from downregulating Pum/Nos function in repressing the translation of differentiation genes and germline Piwi function in promoting germ cell division.     

Drpiwi-1 is essential for germline cell formation during sexualization of the planarian Dugesia ryukyuensis

A piwi homolog is required for the regulation of stem cells, formation and maintenance of germline stem cells, and gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, both asexually and sexually, and develop and maintain germ cells and sexual organs. They have many pluripotent stem cells (neoblasts) that can differentiate into both somatic and germline stem cells. Thus, we searched for a piwi subfamily in the planarian Dugesia ryukyuensis. Four piwi homologs, identified as Drpiwi-1, -2, -3, and -4, were expressed in sexually reproductive worms. We then selectively destroyed the neoblasts by irradiating the worms with X-rays. In such worms, Drpiwi-1, -2, and -3 were not expressed at all, whereas Drpiwi-4 was expressed to the same degree as that in non-irradiated controls, indicating that Drpiwi-1, -2, and -3, but not Drpiwi-4, are expressed in neoblasts. During the regeneration process, Drpiwi-2(RNAi) and -3(RNAi) worms failed to regenerate after ablation, but Drpiwi-1 and -4(RNAi) worms regenerated. During the sexualizing process, Drpiwi-1(RNAi) worms failed to develop ovaries and testes, but somatic sexual organs were unaffected. Germ cell development was normal in Drpiwi-4(RNAi) worms. Therefore, Drpiwi-2 and -3 may be related to the regulation of neoblasts important for maintaining homeostasis, and Drpiwi-1 is essential for the development of germ cells but not somatic sexual organs. DrPiwi-1 is localized in the cytoplasm of stem cells and germline cells and may be involved in regulating some gene expression. We suggest that planarian Piwi controls germline formation via RNA silencing mechanisms.     

Dual functions of Macpiwi1 in transposon silencing and stem cell maintenance in the flatworm Macrostomum lignano

PIWI proteins and piRNA pathways are essential for transposon silencing and some aspects of gene regulation during animal germline development. In contrast to most animal species, some flatworms also express PIWIs and piRNAs in somatic stem cells, where they are required for tissue renewal and regeneration. Here, we have identified and characterized piRNAs and PIWI proteins in the emerging model flatworm Macrostomum lignano. We found that M. lignano encodes at least three PIWI proteins. One of these, Macpiwi1, acts as a key component of the canonical piRNA pathway in the germline and in somatic stem cells. Knockdown of Macpiwi1 dramatically reduces piRNA levels, derepresses transposons, and severely impacts stem cell maintenance. Knockdown of the piRNA biogenesis factor Macvasa caused an even greater reduction in piRNA levels with a corresponding increase in transposons. Yet, in Macvasa knockdown animals, we detected no major impact on stem cell self-renewal. These results may suggest stem cell maintenance functions of PIWI proteins in flatworms that are distinguishable from their impact on transposons and that might function independently of what are considered canonical piRNA populations.     

Piwi Genes Are Dispensable for Normal Hematopoiesis in Mice

Hematopoietic stem cells (HSC) must engage in a life-long balance between self-renewal and differentiation to sustain hematopoiesis. The highly conserved PIWI protein family regulates proliferative states of stem cells and their progeny in diverse organisms. A Human piwi gene (for clarity, the non-italicized “piwi” refers to the gene subfamily), HIWI (PIWIL1), is expressed in CD34⁺ stem/progenitor cells and transient expression of HIWI in a human leukemia cell line drastically reduces cell proliferation, implying the potential function of these proteins in hematopoiesis. Here, we report that one of the three piwi genes in mice, Miwi2 (Piwil4), is expressed in primitive hematopoetic cell types within the bone marrow. Mice with a global deletion of all three piwi genes, Miwi, Mili, and Miwi2, are able to maintain long-term hematopoiesis with no observable effect on the homeostatic HSC compartment in adult mice. The PIWI-deficient hematopoetic cells are capable of normal lineage reconstitution after competitive transplantation. We further show that the three piwi genes are dispensable during hematopoietic recovery after myeloablative stress by 5-FU. Collectively, our data suggest that the function of the piwi gene subfamily is not required for normal adult hematopoiesis.     

The role of PIWI and the miRNA machinery in Drosophila germline determination

BACKGROUND: The germ plasm has long been demonstrated to be necessary and sufficient for germline determination, with translational regulation playing a key role in the process. Beyond this, little is known about molecular activities underlying germline determination. RESULTS: We report the function of Drosophila PIWI, DICER-1, and dFMRP (Fragile X Mental Retardation Protein) in germline determination. PIWI is a maternal component of the polar granule, a germ-plasm-specific organelle essential for germline specification. Depleting maternal PIWI does not affect OSK or VASA expression or abdominal patterning but leads to failure in pole-plasm maintenance and primordial-germ-cell (PGC) formation, whereas doubling and tripling the maternal piwi dose increases OSK and VASA levels correspondingly and doubles and triples the number of PGCs, respectively. Moreover, PIWI forms a complex with dFMRP and DICER-1, but not with DICER-2, in polar-granule-enriched fractions. Depleting DICER-1, but not DICER-2, also leads to a severe pole-plasm defect and a reduced PGC number. These effects are also seen, albeit to a lesser extent, for dFMRP, another component of the miRISC complex. CONCLUSIONS: Because DICER-1 is required for the miRNA pathway and DICER-2 is required for the siRNA pathway yet neither is required for the rasiRNA pathway, our data implicate a crucial role of the PIWI-mediated miRNA pathway in regulating the levels of OSK, VASA, and possibly other genes involved in germline determination in Drosophila.     

Multifunctionality of PIWI proteins in control of germline stem cell fate

PIWI proteins interacting with specific type of small RNAs (piRNAs) repress transposable elements in animals. Besides, they have been shown to participate in various cellular processes: in the regulation of heterochromatin formation including telomere structures, in the control of translation and the cell cycle, and in DNA rearrangements. PIWI proteins were first identified by their roles in the self-renewal of germline stem cells. PIWI protein functions are not limited to gonadogenesis, but the role in determining the fate of stem cells is their specific feature conserved throughout the evolution of animals. Molecular mechanisms underlying these processes are far from being understood. This review focuses on the role of PIWI proteins in the control of maintenance and proliferation of germinal stem cells and its relation to the known function of PIWI in transposon repression.     

The division of Drosophila germline stem cells and their precursors requires a specific cyclin

A fundamental yet essentially unexplored question in stem cell biology is whether the stem cell cycle has specific features. Three B-cyclins in Drosophila, Cyclins (Cyc) A, B, and B3, associate with CDK1 and play partially redundant roles in embryogenic mitosis . Here, we show that the division of Drosophila GSCs and their precursors, the primordial germ cells (PGCs), specifically requires CycB. CycB is ubiquitously expressed in both germline and somatic lineages. However, CycB mutation does not have obvious effect on somatic development but causes PGCs to severely under proliferate. Moreover, both female and male CycB mutant GSCs fail to be maintained properly. Removing Cyclin B specifically from female GSCs causes the same defect, confirming the direct and cell-autonomous function of Cyclin B for GSC division. In contrast, two other G2 cyclins, CycA and CycB3, are also expressed in PGCs and GSCs, but overexpressing CycA cannot rescue the CycB mutant defects. These results indicate that the requirement of CycB for PGC and GSC divisions unlikely reflects the insufficient level of G2 cyclins in the CycB mutant but is in favor of a distinct function of CycB in these cells. Our results indicate that stem cells may use specific cell cycle regulators for their division.     

Two mouse piwi-related genes: miwi and mili

Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. We cloned mouse homologues of piwi by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologues MIWI and MILI showed that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili were found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. Our data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production.     

Drosophila dMyc is required for ovary cell growth and endoreplication

Although the Myc oncogene has long been known to play a role in many human cancers, the mechanisms that mediate its effects in both normal cells and cancer cells are not fully understood. We have initiated a genetic analysis of the Drosophila homolog of the Myc oncoprotein (dMyc), which is encoded by the dm locus. We carried out mosaic analysis to elucidate the functions of dMyc in the germline and somatic cells of the ovary during oogenesis, a process that involves cell proliferation, differentiation and growth. Germline and somatic follicle cells mutant for dm exhibit a profound decrease in their ability to grow and to carry out endoreplication, a modified cell cycle in which DNA replication occurs in the absence of cell division. In contrast to its dramatic effects on growth and endoreplication, dMyc is dispensable for the mitotic division cycles of both germline and somatic components of the ovary. Surprisingly, despite their impaired ability to endoreplicate, dm mutant follicle cells appeared to carry out chorion gene amplification normally. Furthermore, in germline cysts in which the dm mutant cells comprised only a subset of the 16-cell cluster, we observed strictly cell-autonomous growth defects. However, in cases in which the entire germline cyst or the whole follicular epithelium was mutant for dm, the growth of the entire follicle, including the wild-type cells, was delayed. This observation indicates the existence of a signaling mechanism that acts to coordinate the growth rates of the germline and somatic components of the follicle. In summary, dMyc plays an essential role in promoting the rapid growth that must occur in both the germline and the surrounding follicle cells for oogenesis to proceed.     

PIWI Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells

PIWI proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the PIWI protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iPS cells are very similar to littermate control iPS cells. These results indicate that PIWI proteins are dispensable for direct reprogramming of mouse fibroblasts.     

A conserved germline multipotency program

The germline of multicellular animals is segregated from somatic tissues, which is an essential developmental process for the next generation. Although certain ecdysozoans and chordates segregate their germline during embryogenesis, animals from other taxa segregate their germline after embryogenesis from multipotent progenitor cells. An overlapping set of genes, including vasa, nanos and piwi, operate in both multipotent precursors and in the germline. As we propose here, this conservation implies the existence of an underlying germline multipotency program in these cell types that has a previously underappreciated and conserved function in maintaining multipotency.